DESCRIPTION(adapted from applicant's abstract): This renewal application is to continue a fruitful investigation of positively-charged lipids (triesters of the natural product, phosphatidyicholine) as vehicles of gene delivery to cells. The project was successful in meeting its original goals as well as revealing previously unrecognized but important aspects of liposome-mediated gene delivery. Phosphatidylcholine triesters were synthesized for the first time and found to have a host of useful properties in addition to the originally proposed application of delivering DNA to cells. These compounds exhibited low toxicity, remained efficient transfection agents after incubation in serum, required no co-lipids, and were metabolized at a rate appropriate for use as a gene or drug delivery vehicle in vivo. Variations in chemical structure led to derivatives with widely different properties. All liquid-crystalline versions generated effective transfection agents, provided formulation conditions were carefully optimized. The standard formulation of the complexes of prototype lipid and DNA generated a multi-decker sandwich structure with DNA and lipid in alternating layers. Development of sophisticated methods of studying the structure of the complex by titration calorimetry, flow cytometry and several spectrofluorometric procedures revealed that its process of formation is more complicated than generally appreciated and variation in formulation procedures greatly influence both the structure and transfection activity of a given lipid. Hence, to progress to clinical uses of cationic lipids, it is essential to elucidate the relationship between formulation conditions, lipid-DNA complex structure, stability under in vivo conditions and transfection efficiency. Accordingly, the specific aims of the renewal application are: 1. To build on our experience with the first generation compounds by synthesizing a limited number of new cationic phospholipids with specific properties to enhance transfection. 2. To understand how formulation conditions affect the structure, stability in serum and ability to modify for targeting, the transfection complexes of these and a select group of other important cationic lipids. 3. To relate those properties of the transfection complexes to transfection efficiency under conditions relevant in vitro and in vivo (including targeting of the complex to specific cell surface markers). 4. To expand collaborations to facilitate development of in vivo applications that should become important clinically.